Project Summary Significant advances in our understanding of the in vivo functions of human cells and tissues especially the human immune system have resulted from the development of ?humanized? mouse models, defined as immunodeficient mice that have been engrafted with human hematopoietic stem cells and/or tissues that generate a functional human immune system. Realizing that large animal species often have an enhanced ability to predict clinical outcome relative to mice, we worked to develop immunodeficient rabbit models using CRISPR/Cas9 and have generated four lines of immunodeficient rabbits: (i) Foxn1 KO; (ii) Prkdc KO; (iii) Rag2 KO; and (iv) Il2rg KO. In the present project, we propose to develop humanized rabbit models by engrafting of human hematopoietic stem (hCD34+) cells in immunodeficient rabbits. These humanized rabbits are useful model to study immunology, regenerative medicine, and are expected to facilitate therapy development for many human diseases especially infectious diseases. In Aim 1, we will breed Rag2 and Il2rg KO rabbit (both on the New Zealand White background) to establish RG (Rag2-/-, Il2rg null) rabbit line. In Aim 2, RG rabbits will be engrafted with human hematopoietic stem cells to assess human immune system development and function. This is a multiple PI project. Dr. Jie Xu at the University of Michigan produced all immunodeficient rabbits. Dr. Moses Bility at the University of Pittsburgh is an expert on developing humanized mouse models. The propose work, if successful, will provide for the first time a humanized large animal model for biomedical research.